Cloned Babesia DNA

ABSTRACT

The subject invention concerns the identification of novel merozoite surface proteins of Babesia bovis. Also disclosed are monoclonal antibodies to these proteins as well as genes which encode for the proteins. The invention further concerns the use of the novel proteins, recombinant DNA clones, and monoclonal antibodies in the detection, treatment, and prophylaxis of babesiosis.

This is a continuation application under 37 CFR 1.53 of the applicationSer. No. 07/989,616 filed Dec. 14, 1992, now abandoned, which is adivisional application of Ser. No. 07/504,461, filed Apr. 4, 1990, nowU.S. Pat. No. 5,171,685, issued Dec. 15, 1992, which is acontinuation-in-part of application Ser. No. 07/333,155, filed Apr. 4,1989, abandoned.

BACKGROUND OF THE INVENTION

Bovine babesiosis is a tick-transmitted, hemoparasitic disease caused byintraerythrocytic protozoa belonging to the genus Babesia. The diseasecaused by Babesia manifests itself clinically by fever and extensivehemolytic anemia that often leads to hypotensive shock, cerebralinvolvement, and death. More than a half billion cattle are estimated tobe at risk of acquiring babesiosis. This disease represents a primaryimpediment to food and fiber production in much of the world.

To date, control of bovine babesiosis in enzootic areas has beenpartially successful through vaccination with attenuated strains ofBabesia spp. or with more virulent strains followed by chemotherapeuticcontrol. Protective immunity in babesiosis may be directed against oneor more surface antigens associated with sporozoites, infectederythrocytes, and/or merozoites. Merozoite surface antigens areimportant in the pathogenesis and immunology of babesiosis due to theirrole in the parasite's recognition of, attachment to, and penetration ofhost erythrocytes and their accessibility to the immune system.

Recently, progress has been made toward the identification andcharacterization of specific immunogens of merozoites of Babesia bovis(Smith, R. D., M. A. James, M. Ristic, M. Aikawa, and C. A. Vega YMurgula [1981] Science 212:335-338; Wright, I. G., B. V. Goodger, K.Rode-Bramanis, J. S. Matlick, D. F. Mahoney, and D. J. Waltisbuhl [1983]Z. Parasitenkd. 69:703-714; Wright, I. G., G. B. Mirre, K.Rode-Bramanis, M. Chamberlain, B. V. Goodger, and D. J. Mahoney [1985]Infect. Immun. 48:109-113; Commins, M. A., B. V. Goodger, and I. G.Wright [1985] Int. J. Parasitol. 15:491-495; Wright, I. G. and P. W.Riddles [1986] "Biotechnological Control of Tick-Borne Disease," Meetingof the Food and Agriculture Organization of the United Nations, 6-10Oct. 1986, pp. 1-21, Rome, Italy; Waltisbuhl, D. J., B. V. Goodger, I.G. Wright, G. B. Mirre, and M. A. Commins [1987] Parasitol Res.73:319-323; Goff, W. L., W. C. Davis, G. H. Palmer, and T. C. McGuire[1988] Infect. Immun. 56:2363-2368) and Babesia bigemina (McElwain, T.F., L. F. Perryman, W. C. Davis, and T. C. McGuire [1987] J. Immunol.138(7):2298-2304). However, in only one instance (Smith et at., 1981)were antigens which provided protection against infection determined tobe surface-exposed on merozoites as opposed to cytoplasmic in location.

Bovine babesiosis can be caused by either Babesia bigemina or Babesiabovis. These parasites have antigenic similarities and differences thatmay have important functional roles in the induction of protectiveimmunity and antibody-based diagnosis. Also, B. bovis isolates,including the current Australian vaccine strain, are now known toconsist of subpopulations that vary antigenically, in virulence, and inabundance within an isolate (Cowman, A. F., P. Timms, and D. J. Kemp[1984] Mol. Biochem. Parasitol. 11:91-103; Gill, A. C., A. F. Cowman, N.P. Stewart, D. J. Kemp, and P. Timms [1987] Exp. Parasitol. 63:180-188).

Current vaccine strategies include the use of attenuated live Babesiabovis parasites and various inactivated preparations (Montenegro-James,S., M. Toro Benitez, E. Leon, R. Lopez, and M. Ristic [1987] Parasitol.Res. 74:142-150; Smith et al., 1981; U.S. Pat. No.4,762,711 issued toBuening et at.; Kuttler, K. L., M. G. Levy, M. A. James, M. Ristic[1982] Am. J. Vet. Res. 43(2):281-284). The attenuated vaccine providesthe best protection against challenge with both homologous andheterologous strains, although there are a number of seriousdisadvantages, including a short shelf-life, variation in virulence,contamination with host erythrocyte stroma, and perpetuation of the lifecycle by creation of a carrier state. Inactivated vaccines induceprotection against challenge with homologous strains; however, onlypartial protection occurs against challenge with heterologous strains.

Animals that survive natural field infection or that recover frominfection with an attenuated vaccine strain are protected againstclinical disease. However, premunization in this manner is expensive,impractical in developing countries that lack the necessaryinfrastructure, and a potential mode of transmission for otherblood-borne diseases.

BRIEF SUMMARY OF THE INVENTION

Disclosed and claimed here are novel merozoite proteins of Babesiabovis. These proteins are known to be expressed on the surface of themerozoite and may be used to raise neutralizing antibodies. Thus, theycan be used in the formulation of subunit vaccines for the prophylaxisof bovine babesiosis. Several of the proteins described here raiseantibodies to both Babesia bovis and Babesia bigemina, while others arespecies, or even isolate, specific.

Also disclosed are monoclonal antibodies to bovine babesiosis antigens.These monoclonal antibodies are used to identify merozoite surfaceantigens and may be used in the treatment and/or diagnosis of bovinebabesiosis.

A further element of the invention is the identification of genes whichcode for Babesia proteins. These genes can be used to make recombinantproteins which can be utilized for vaccines.

The invention also provides a means of detecting the presence ofdisease-causing Babesia organisms. The detection method involves the useof DNA probes which selectively identify the presence of theseorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is the DNA sequence of lambda-Bo44.

FIG. 1B is the amino acid sequence of lambda-Bo44 in the single-letterdesignations.

FIG. 2A is the DNA sequence for rBv42.

FIG. 2B is the amino acid sequence for rBv42 in the single-letterdesignations.

FIG. 3-1 through 3-2 is the DNA sequence for rBv60.

FIG. 4 is the amino acid sequence for rBv60 in the single-letterdesignations.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention pertains to the identification of surface-exposedproteins of B. bovis merozoites. The proteins of the invention havesizes of 16, 25, 37, 42, 44, 55, 60, 85, 98, 125, 145, 225, and 250 kDa.The evidence that the proteins are surface exposed includes: (i)monoclonal antibody binding of live merozoites, (ii) labeling by surfaceiodination, and (iii) sensitivity to mild trypsinization.

We have identified B. bovis merozoite proteins that, by virtue of theirsurface location and their reactivity with immune bovine sera, arecandidates for subunit vaccines. Among the numerous proteins recognizedby immune bovine sera, six proteins (37, 42, 55, 85, 125, and 145 kDa)appeared to be relatively immunodominant.

The 145 kDa protein was of parasite origin, but its location on themembrane surface was not directly apparent. This protein may have asmall portion exposed at the surface of the merozoite that is sensitiveto mild trypsinization but the epitope recognized by the monoclonalantibody located internally.

Immunoprecipitation of radiolabeled antigens with bovine antiseraindicated that many Babesia bovis merozoite proteins containisolate-common epitopes, while at least 8 B. bovis proteins containspecies-cross-reactive epitopes. The amino acid sequence of three of theimmunogenic proteins from B. bovis (42 kDa) have been determined. Aminoacid sequences which deviate in insignificant ways from the disclosedamino acid sequences fall within the scope of the subject invention solong as the antigenic properties of the protein are not altered. Thus,the subject invention includes mutants and fragments of the amino acidsequences depicted herein which do not alter the protein secondarystructure, or if the structure is altered, the antigenic activity isretained. In particular, it should be understood that conservativesubstitutions of amino acids may be made. For example, amino acids maybe placed in the following classes: basic, hydrophobic, acidic, polar,and amide. Substitutions whereby an amino acid of one class is replacedwith another amino acid of the same type fall within the scope of thesubject invention so long as the substitution does not materially alterthe antigenic activity of the compound.

The ability of antibodies against heterologous geographic isolates toimmunoprecipitate proteins from the Mexico B. bovis isolate indicatesthe conservation of at least one and probably more epitopes betweenproteins from the heterologous isolates. The conservation of theseepitopes is extensive, as many Mexico B. bovis isolate proteins wereprecipitated by antisera against a different geographic isolate(Honduras). The 42,000 molecular weight Mexico B. bovis protein wasprecipitated by all five of the undiluted and three of the dilutedHonduras antisera.

Among the highly immunogenic B. bovis proteins, only one (the 42,000molecular weight protein) is both isolate common and species specific.This protein can be used as an antigen for species-specific,antibody-based diagnosis.

Monoclonal antibodies (MoAbs) were generated against surface-exposedproteins on merozoites of B. bovis. A genomic library constructed in thelambda-gt11 expression vector was screened with MoAbs for identificationof clones expressing recombinant surface proteins. Four recombinantclones were identified.

Southern blot analyses confirmed the parasite-specificity of the clonedinserts. Western blot analyses demonstrated that recombinant proteinproduction in these clones is IPTG-induced and that the recombinantmolecules exist as beta-galactosidase fusion proteins.

Additionally, recombinant proteins, partially purified by affinitycolumn chromatography and gel filtration chromatography, reacted withspecific MoAbs in Western blot assay indicating that the integrity ofthe epitopes is retained during purification. Calves immunized withthese partially purified recombinant proteins developed titers ofbetween 10⁻² and 10⁻⁵ as evidenced by IFA-live. Immune sera from theseanimals immunoprecipitated metabolically-radiolabeled merozoite proteinsconfirming that determinants found on native proteins are expressed bythe clones.

DNA probe candidates were also identified using the lambda-gt11 genomiclibrary of B. bovis. Two DNA sequences, designated lambda-Bo6 andlambda-Bo25, hybridized to Babesia DNA but not to bovine DNA. Bo6detected Mexico and Australia isolates of B. bovis as well as B.bigemina DNA. Lambda-Bo25 demonstrated greater specificity; it did nothybridize detectably to B. bigemina DNA and showed greater sensitivityfor Mexico isolates of B. bovis than for Australia isolates.

Thus, lambda-Bo6 is a good candidate for detecting Babesia infections incattle and ticks, and lambda-Bo25 can be used to distinguish geographicisolates of B. bovis.

Materials and Methods

Strain of B. bovis, Stabilate Preparation, Cryopreservation, and InVitro Cultures. The strain of B. bovis used in experiments outlinedherein was originally isolated from a Boophilus microplus tick-inducedinfection in Mexico by Dr. R. D. Smith, University of Illinois at Urbana(Goff, W. L. and C. E. Yunker [1986] Exp. Parasitol. 62:202-210). Thecloned line was derived from the Mexico isolate by limiting dilutioncloning as previously described by Rodriguez et al. (Rodriguez, S. D.,G. M. Buening, T. J. Green, and C. A. Carson [1986] Infect. Immun.42:15-18). The parasites have been maintained in our laboratory byeither repeated passages in splenectomized Holstein-Freisian bull calvesor in vitro cultivation. Cryopreservation of stabilates of B.bovis-infected erythrocytes obtained from infected calves and thepreparation of partially purified merozoites from thawed stabilates hasbeen described (Palmer, D. A., G. M. Buening, and C. A. Carson [1982]Parasitol 84:567; McElwain, T. F., L. E. Perryman, W. C. Davis, and T.C. McGuire [1987] J. Immunol. 138:2298-2304 ). Continuous in vitrocultivation of B. bovis was performed using a modification of themicroaerophilous stationary phase (MASP) culturing system (Goff andYunker, 1986). Viability of merozoites obtained from either frozenstabilates or in vitro cultivation was confirmed by 6-CFDA assay(McElwain et al., 1987) prior to their use in experiments orimmunizations.

Isolation of Merozoites. Merozoites were harvested from cultures afterthe relative percentage of parasitized erythrocytes was increased bysequential reduction of the concentration of erythrocytes (Goff andYunker, 1986). For collection of merozoites, the contents of flaskscontaining >15% parasitized erythrocytes were centrifuged at 400×g for10 min at 4° C. The supernatant was centrifuged at 3,000×g for 15 min at4° C. to pellet the merozoites. The merozoites were suspended in Pucksaline-glucose (saline-G), and 2 ml was overlaid on 10 ml of a preformedcontinuous gradient of 65% Percoil-35% Puck saline-G. The gradient wascentrifuged in a swinging bucket rotor at 3,000×g for 20 min at 4° C.The merozoites were isolated from a band with an approximate density of1.069 g/ml between erythrocyte ghosts at the Percoil-Puck saline-Ginterface and the residual intact erythrocyte pellet. The merozoiteswere washed once in 0.15M NaCl containing 0.01M sodium citrate (CS),suspended in CS, and stored on ice until used (within 2 to 4 hr).

Purification, Quantitation and Viability Estimation of Merozoites. Anequal volume of the isolated merozoite suspension was mixed with6-carboxy fluorescein diacetate (6-CFDA; final concentration in CS, 10ug/ml; Calbiochem-Behring, La Jolla, Calif.) (McElwain et al., 1987).The mixture was incubated at room temperature for 20 min, followed bycentrifugation at 1,000×g for 10 min and was then suspended inphosphate-buffered saline (PBS; 0.15M, pH 7.2) for counting on ahemacytometer. The sample was examined with phase microscopy andepifluorescence with a 40X oil objective and fluorescein filter (450 to520 nm). Viability was assessed as the percentage of total merozoitesemitting fluorescence.

Preparation of Immune Bovine Sera. Two spleen-intact Holstein-Freisiansteers, 14 months of age and indirect fluorescent-antibody test negativefor B. bovis, B. bigemina, and Anaplasma marginale, were inoculatedintravenously with approximately 6×10⁸ B. bovis-infected erythrocytesfrom the same blood stabilate used to initiate in vitro cultures. On day9 postinoculation each steer developed detectable parasitemia and afebrile response which persisted through day 13 postinoculation.Antibody specific for B. bovis was detected with the indirectfluorescent-antibody test on day 10 postinoculation. The steers werechallenge inoculated as before on days 48 and 80 postinoculation, andalthough the animals did not develop a fever or parasitemia, theantibody titer increased after each challenge. Sera were collected andstored at -70° C. after the final challenge, when the indirectfluorescent-antibody test tiler was 1:10,000.

In addition, the cloned line was passed through a splenectomized calfwhose blood at peak parasitemia was used to infect five 4-5 month oldHolstein steers (5×10⁷ infected erythrocytes each) and to initiate invitro cultures. The cattle were reinfected at 23 days post infection(DPI) with 10⁸ infected erythrones (iRBC) from another splenectomizedcalf and at 77 and 99 DPI with 10⁸ iRBC from culture. At day 127, thefive cattle and three weight-matched, previously uninfected controlswere infected with 10⁹ iRBC from culture. The packed cell volume (PCV)of all animals was monitored daily.

Immunofluorescence of Live Merozoites. Viable merozoites were collectedas described above and reacted with the various antibodies by apreviously described technique (McElwain et al. [1987], supra). TheMoAb-containing ascites fluids were diluted 1/10 (40 ug/ml) in PBS.Immune bovine sera were diluted 1/10 in PBS. An equal volume of eachantibody preparation was added to 100 ul of a merozoite suspension andincubated on ice for 1 hr. Each sample was centrifuged at 3,000×g for 10min, and the merozoites were washed twice in cold PBS. The samples werethen suspended in the appropriate rhodamine-conjugated second antibody(1/40 dilution in PBS) (Kirkegaard and Perry, Inc., Gaithersburg, Md.)and incubated on ice for 1 hr. After being washed, the merozoites weresuspended in 6-CFDA and incubated for 20 min at room temperature. Themerozoites were then centrifuged at 3,000×g for 10 min at 4° C.,suspended in 50 ul of PBS, and examined in wet mounts with appropriatefilters for rhodamine (antibody binding) and fluorescein (6-CFDAviability) (546 to 590 nm and 450 to 520 nm, respectively).

Surface Radioiodination. Purity of the gradient separated merozoites wasalso examined by direct light microscopy of Giemsa stained smears and bytransmission electron microscopy of selected samples fixed in 2% v/vglutaraldehyde in 0.1M potassium phosphate buffer containing 1% w/vsucrose. Parasites were often arranged in clumps and mixed with veryrare erythrocyte ghosts (<0.1%). Merozoites were surface radioiodinatedby a previously described lactoperoxidase catalyzed method (Palmer, G.H., and T. C. McGuire [1984] J. Immunol. 133:1010-1015).

Donor erythrocytes (nRBC) from uninfected control cultures werecollected, washed three times in PBS, and radiolabeled identically. Anequivalent number of nRBC ghosts were prepared by lysing washeduninfected cells from control cultures by freeze/thaw in liquidnitrogen. Ghosts were washed free of hemoglobin in PBS by centrifugationat 35,000×g, 20 min, 4° C. and discarding the supernatant until it wasclear. The final pellet was resuspended in PBS for radioiodination bylactoperoxidase.

Metabolic Radiolabeling of Merozoites. Metabolically radiolabeledparasite proteins from calf-derived merozoites were prepared for use inimmunoprecipitation experiments according to the methods of McElwain etal. (1987, supra) except that cultures containing 100 uCi of [³⁵S]-methionine (³⁵ S-Met; New England Nuclear, Boston, Mass.) per 3×10⁹erythrocytes were incubated at 37° C. for 8-9 hr in a Forma Scientificwater jacketed incubator instead of a candle jar. Parasites cultivatedin vitro were metabolically radiolabeled using normal growth medium(Goff and Yunker, 1986) or D,L-methionine-free medium, addition of20-400 uCi/ml ³⁵ S-Met, ³ H-myristic acid, or ³ H-glucosamine, andincubation of cultures for 12-20 hr at 37° C. Erythrocytes containingradiolabeled merozoites were solubilized in lysis buffer,TCA-precipitable radioactive counts were determined by a filter papertechnique (New England Nuclear), and samples were frozen at -70° C.until used.

Phase Separation in TRITON™X-114. Washed iRBC's from ³⁵ S-methioninelabeled cultures were lysed in 10 mM Tris, 154 mM NaCl pH 7.4, 1% (v/v)TRITONT™X-114, 1 mM phenylmethylsulfonyl fluoride (PMSF) at 0°-4° C. andfrozen at -20° C. For protein separation, the antigen extract was firstprocessed as described for immunoprecipitation. 10⁷ protein bound countsper minute (CPM) in a volume of 2.0 ml was laid over a 2.0 ml cushion of6% (w/v) sucrose, 10 mM Tris, 154 mM NaCl, 1 mM PMSF in a 15 ml conicaltube (Bordier, C. [1981] Exp. Parasitol. 20:125-129). The tube wasincubated at 37° C. for 5 min to allow clouding of the protein extractand then centrifuged at 750×g at room temperature for 5 min in aswinging bucket rotor. The detergent phase was seen as a thick, oily100-200 ul pellet and the overlying aqueous phase and sucrose cushionwere each removed to separate tubes. The phase separation was repeatedtwice by adding 200 ul of 15% (v/v) TRITON™X-100 in 10 mM Tris, 154 mMNaCl to the aqueous phase, dissolving the detergent on ice, andre-extracting at 37° C. over the same sucrose cushion. The threedetergent phases resulting from centrifugation were mixed with 10 mMTris, 154 mM NaCl at 0°-4° C., combined, and TCA-precipitableradioactivity counted along with the aqueous phase.

Immunoprecipitation. Immune sera were used either unadsorbed or adsorbedthree times with an equal volume of packed intact nRBC's and three timeswith an equal volume of nRBC ghosts. Radiolabeled B. bovis or bacteriallysate was processed as described previously (Palmer and McGuire [1984],Supra) and incubated overnight at 4° C. with 15 ul of bovine serum or 15ul of serum diluted in Veronal buffered saline (VBS) pH 7.4, 1% (v/v)Nonidet P-40 (NP-40). 150 ul of 10% (v/v) formalinized Protein G-bearingStreptococcus (Omnisorb, Calbiochem, San Diego, Calif.) in VBS pH 7.4,1% (v/v) NP-40, 0.1% (w/v) gelatin was added and incubated for 2 hr at4° C. (Akerstrom, B., T. Brodin, K. Reis, and L. Bjorck [1985] J.Immunol. 135:2589-2592). The precipitates were washed twice with VBS, 1%(v/v) NP-40; four times with VBS, 2M NaCl, 1% (v/v) NP-40, 10 mMethylenediaminetetraacetic acid (EDTA); and twice more with VBS, 1%(v/v) NP-40. Alternatively, 5 ul of bovine serum, 10 ul of rabbit serum,or 5 ug monoclonal antibody were incubated for 30 minutes at 4° C. withradiolabeled lysate. Rabbit anti-bovine or rabbit anti-mouseimmunoglobulin sera were added and incubated for 30 minutes at 4° C.,followed by 10% v/v protein-A-bearing Staphylococcal aureus for 30minutes at 4° C. Immune complexes were washed seven times with TENbuffer (20 mM Tris-HCl, 5 mM EDTA, 0.1 mM NaCl, 15 mM NAN₃, pH 7.6)containing Nonidet P-40, and for the second through fifth washes, 2MNaCl, by centrifuging at 1250×g. The precipitated protein was eluted bya described method and either frozen at -20° C. or loaded directly ontoa polyacrylamide gel (Palmer and McGuire [1984], supra).

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) ofImmunoprecipitates and Autoradiography. Immunoprecipitates wereelectrophoresed under reducing conditions in 7.5 to 17.5% continuousgradient polyacrylamide gels (Takac, B. [1979] In: ImmunologicalMethods. T. Lefkovitz and B. Persin, eds., p. 81, Academic Press, NewYork). ¹⁴ C-labeled protein standards used for molecular weightdetermination were myosin, 200 kDa; phosphorylase b, 92.5 kDa; bovineserum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa;and lysozyme, 14.3 kDa (Amersham Corp., Arlington Heights, Ill.). For¹²⁵ I-labeled antigens, gels were fixed in 30% (v/v) methanol, 10% (v/v)acetic acid, vacuum dried, and exposed to Kodak XAR-2 X-ray film with anintensifying screen at -70° C. For ³ H and ³⁵ S-labeledimmunoprecipitates, gels fixed in 30% (v/v) methanol, 10% (v/v) aceticacid, 10% (w/v) trichloroacetic acid (TCA) were impregnated with En³Hance (New England Nuclear Corp., Boston, Mass.) prior to drying andexposure to X-ray film at -70° C. (Palmer and McGuire, 1984).

Immunoblotting. Immunoblotting of merozoite and recombinant lysogenproteins using monoclonal antibodies was performed as outlined (McElwainet at., 1987) using standard procedures (Towbin, H. and J. Gordon [1984]J. Immunol. Methods 72:313-340). Parasite antigen for immunoblotting wasprepared from MASP culture flasks with approximately 25% parasitemia.Briefly iRBC's and nRBC controls were collected, washed two times incold Puck's saline-G and two times in cold PBS, resuspended in PBS,counted, and frozen at -20° C. To remove hemoglobin from lysed cells,the samples were thawed and washed in cold PBS (43,000×g, 20 min, 4° C.)until the discarded supernatant was clear. The final pellet wasextracted in lysis buffer, processed identically to radiolabeled antigenfor immunoprecipitation, and aliquoted for freezing at -20° C. A volumecorresponding to 2.5×10⁷ iRBC's or an equivalent total number of nRBC's(10⁸ ) was mixed with 3× SDS-PAGE sample buffer, boiled for 3 minutes,electrophoresed in a 7.5 to 17.5% continuous gradient polyacrylamidegel, and then electrophoretically transferred overnight to anitrocellulose membrane (Towbin and Gordon, 1984). Immunoblotting usingbovine antisera was performed as follows: The nitrocellulose was washedthree times quickly in VBS pH 7.4, 0.25% (v/v) TWEEN™20, 0.25% (w/v)gelatin (blocking buffer), incubated 4-6 hr in blocking buffer, cut intostrips, and each strip reacted overnight at room temperature with immuneserum diluted in blocking buffer. The nitrocellulose strips were thenwashed three times in blocking buffer and two times in VBS 0.1% (w/v)gelatin prior to incubation for 2 hours at room temperature with ¹²⁵I-Protein G (Amersham Corp.) in VBS pH 7.4, 0.1% (w/v) gelatin(Akerstrom, B., T. Brodin, K. Reis, and L. Bjorck [1985] J. Immunol.135:2589-2592). The strips were washed twice with VBS 0.1% (w/v) gelatinand four times with 1M NaCl, 10 mM EDTA, 0.25% (v/v) TWEEN™20. They werethen air-dried, taped to cardboard, and exposed to X-ray film with anintensifying screen at -70° C.

Dot Blot Immunoassay. Because MoAbs were used to screen the lambda-gt11genomic library for clones expressing recombinant surface proteins, theywere first evaluated for their ability to bind native antigen applied tonitrocellulose filters. 6-CFDA-positive merozoites were obtained fromfrozen blood stabilates, lysed in buffer containing 50 mM Tris, 5 mMEDTA, 5 mM iodoacetamide, 1 mM PMSF, 0.1 mM N-alpha-p-tosyl-L-lysylchloromethyl ketone (TLCK) and 1% NP-40 (lysis buffer), and frozen at-70° C. until use. Aliquots of 1 ul containing either 10⁷, 10⁶ or 10⁵merozoites were spotted onto nitrocellulose filters and air dried.Corresponding numbers of similarly lysed noninfectedbovine erythrocyteswere spotted on for control. Nitrocellulose filters with spotted antigenwere washed three times (10 min each) in buffer containing 10 mM Tris(pH 8.0), 150 mM NaCl, 0.05% TWEEN™20, and 0.1 mM PMSF (TNTP) thenincubated in TNTP with 5% nonfat dry milk for 1 hr to block unboundsites. Filters were washed three times in TNTP plus 5% milk, incubatedin the same buffer containing 2 ug/ml of specific surface-binding MoAb(1/2 hr), washed three times, incubated for 1/2 hr in a 1:5000 dilutionof rabbit anti-mouse immunoglobulin (prepared in our laboratory) in TNTPplus 5% milk. After three washes, the filters were incubated for 1/2 hrin TNTP plus 5% milk containing 5×10⁶ CPM of ¹²⁵ I-labeled Protein A,washed sequentially with TNTP, TNTP plus 0.1% TRITON™X, and TNTP, thendried and examined by autoradiography.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Generation of Monoclonal Antibodies Against Surface Epitopes

Partially purified merozoites of B. bovis obtained from frozen bloodstabilates were used to immunize BALB/c mice for hybridoma production.Each mouse received an initial subcutaneous immunization of 10⁷6-CFDA-positive organisms in Freund's complete adjuvant followed by 3-4subcutaneous immunizations of 10⁷ 6-CFDA-positive organisms in Freund'sincomplete adjuvant at 2-4 week intervals. Mouse serum was titered byIFA-live (Barbet, A. F. and T. C. McGuire [1978] Proc. Natl. Acad. Sci.USA 75:1989-1993) after the last immunization, and mice with high titers(≧1:1000) received an intravenous booster immunization of 10⁶6-CFDA-positive organisms in sterile PBS. Three days later the mice werekilled and their spleen cells fused to SP2/0 myeloma cells usingstandard procedures (McGuire, T. C., L. E. Perryman, and W. C. Davis[1983] Amer. J. Vet. Res. 44:1284-1288). Hybridoma supernates werescreened first for convenience by IFA-fixed (Ross, J. P. J and K. F.Lohr [1968] Res. Vet. Sci. 9:557-562). Positive supernates were thenscreened by IFA-live using stabilate-derived merozoites in order toidentify surface reactive MoAbs (McElwain et al. [1987]). The MoAbs werefirst screened on fixed infected erythrocyte preparations, and fourMoAbs were selected for further evaluation because of their distinctivepatterns of fluorescence. These patterns included Staining of themerozoite cytoplasm and membrane (BABB35A₄), merozoite membrane(BABB90C₄), and merozoite cytoplasm (BABB93A₁) and a single, punctatereaction appearing polar in location on merozoites (BABB75). The fourMoAbs all retained their original specificities after the hybridomacells were cloned twice and used to produce ascites fluids.

EXAMPLE 2

Immunoprecipitation of Surface Radioiodinated and MetabolicallyRadiolabeled Proteins

Spontaneously released merozoites for surface binding and labelingexperiments were isolated on Percoil gradients. A large proportionretained their surface coat and >80% were viable, as determined by6-CFDA staining. On three occasions, 10⁸ of these isolated merozoiteswere inoculated intravenously into susceptible, splenectomized calves.In each case, infection was achieved with a prepatent period similar tothat in calves that received an equivalent number of infectederythrocytes from a blood stabilate. Also, in vitro cultures have beenroutinely reestablished after introduction of these isolated merozoites.

Radioiodinated merozoite preparations were immunoprecipitated with theMoAbs described above to confirm the outer surface or cytoplasmiclocation of the reactive epitopes. BABB35A₄ precipitated a major proteinof 42 kDa and a minor protein of 37 kDa. BABB75 and BABB90C₄precipitated single proteins of 60 and 85 kDa, respectively.

To determine which parasite proteins were recognized by the bovineimmune system, we used twofold serial dilutions of immune bovine sera toimmunoprecipitate metabolically labeled preparations. Proteins withrelative molecular masses ranging from approximately 16 to >200 kDa wererecognized by the immune bovine sera. Among the proteins recognized werethose identical in molecular mass to those precipitated by BABB35A₄ (42and 37 kDa), BABB75 (60 kDa), and BABB93A₁ (145 kDa). In addition,proteins of 145, 42, 120 and 75 kDa appeared to be immunodominant, asthey were precipitated by immune bovine sera at the greatest dilutiontested.

Example 3

Further Identification of Merozoite Surface Proteins

Subinoculation of 5×10⁷ infected erythrocytes (iRBC) of a cloned B.bovis line from a splenectomized calf into 4-5 month old cattle caused a39% reduction in packed cell volume (PCV) (range 31-47). Calves werere-infected three times with approximately 10⁸ iRBC of the clonedisolate and then challenged with 10⁹ iRBC in concert with threepreviously uninfected control animals. Only the initial infection causeda significant reduction in PCV when compared to the PCV during the weekprior to each infection. Control cattle in the final challengeexperiment experienced a 28% reduction in PCV (p< or =0.0005 whencompared to previously infected cattle; Student's paired t test).

Merozoites spontaneously released from culture and purified on Percollgradients were 95-100% viable by 6-CFDA staining. In all five animals,immunoprecipitation of surface radioiodinated proteins with immune serathat had been extensively adsorbed against donor erythrocytes (nRBC) anderythrocyte ghosts identified seven dominant surface proteins withrelative molecular weights of 250, 125, 98, 85, 55, 42, and 37kilodaltons. The 250 kDa protein does not enter the resolving gel in astandard 14 cm 7.5-17.5% polyacrylamide gel but is clearly resolved in a25 cm gel. An eighth protein of 25 kDa is immunoprecipitated by immunesera from two calves. Control immunoprecipitation of identicallyradioiodinated intact nRBC and nRBC ghosts revealed no specific bands onSDS-PAGE.

Adsorbed immune sera was used to immunoprecipitate ³⁵ S-methioninemetabolically labeled parasite proteins which were run alongsideimmunoprecipitated surface-iodinated merozoite proteins in apolyacrylamide gel. The immunoprecipitable ³⁵ S antigen profile isidentical in all five protected animals. The 125, 98, 85, 55, 42, and 37kDa antigens comigrate perfectly with metabolically labeled proteins.The 25 kDa surface protein that is not identified by immunoprecipitationof methionine labeled antigen does comigrate with a glycoprotein that ismetabolically labeled with 3H-glucosamine. An ³⁵ S-methionine labeled 25kDa protein can be precipitated from other ³⁵ S-antigen preparations.

EXAMPLE 4

Immunogenicity

While the reactivity of immune sera against the majority of ³⁵ S-labeledproteins can be diluted out, sequential serum dilutions (1:160-1:640)selectively precipitate the 125, 55, and 42 kDa proteins that were alsosurface labeled. Because this method is dependent on the specificradioactivity of labeled proteins, dilute sera was also examined for itsability to react with parasite antigens by immunoblotting. Compared toundiluted serum, immune sera diluted 1:500 recognizes a limited numberof blood stage proteins including the 125, 85, 55, and 42 kDa surfaceantigens. The 42 kDa protein is consistently recognized even atdilutions of greater than or equal to 1:16,000.

EXAMPLE 5

Further Characterization of Proteins

The immunodominant 42 kDa merozoite surface protein was furthercharacterized as an integral membrane protein based on its hydrophobicnature in phase separated TRITON™X-114 solution. By definition, integralmembrane proteins have a hydrophobic domain that allows interaction withthe hydrophobic core of the lipid bilayer and with non-ionic detergents.Parasite proteins were metabolically labeled in culture with ³⁵S-methionine and solubilized in 1% TRITON™X-114 at 0-4° C. The antigenpreparation was warmed above the detergent's cloud point (20° C.) andseparated into aqueous and detergent phases by centrifugation.Immunoprecipitation from each phase and the starting solution shows thatthe 42 kDa antigen partitions into the detergent phase.

In order to better characterize merozoite surface antigens, the parasitewas examined for the ability to incorporate ³ H-glucosamine and ³H-myristic acid into immunoprecipitable proteins. Comigration on apolyaerylamide gel shows that three of the surface labeled proteins (55,42, and 25 kDa) are glycosylated and the 42 kDa glycoprotein ismyristylated.

EXAMPLE 6

Further Studies on Immunogenicity

Antiserum C151, which was used for immunoprecipitations, was collectedfrom a spleen-intact cow 60 days after experimental infection with acryopreserved Mexico isolate blood stabilate of B. bovis. B. bovisproteins were metabolically labeled in microaerophilus stationary-phaseculture by incubation in methionine-deficient medium for 18 to 24 hrwith 10 uCi of [³⁵ S]methionine per ml. Antiserum C151immunoprecipitated homologous Mexico isolate proteins biosyntheticallylabeled with [³⁵ S]methionine with molecular weights ranging from 14,500to greater than 200,000. Serial dilution of this antiserum resulted in adecrease in the number of proteins recognized. Proteins reactive withserum diluted 1:40 had relative molecular weights of 145,000, 120,000(doublet), and 42,000, while the 42,000 molecular weight protein wasstill recognized by serum diluted 1:80.

Example 7

B. bovis Proteins with Isolate-Common Epitopes

Five different antisera obtained from cattle after recovery from acuteinfection with B. bovis in Honduras were able to immunoprecipitate mostof the Mexico isolate B. bovis proteins precipitated by C151 antiserum.The 120,000 and 42,000 molecular weight proteins recognized by 1:40dilutions of C151 antiserum were also recognized by 1:25 dilutions ofthe Honduran antisera.

EXAMPLE 8

B. bovis Proteins with Species-Common Epitopes

Antiserum B85 was collected from a spleen-intact Calf 25 days afterexperimental infection with a cryopreserved Mexico isolate of B.bigemina. This antiserum, which had an indirect fluorescent-antibodytiter of 1:1,600 against the Mexico B. bigemina isolate, reacted withthe Mexico B. bovis isolate at a titer of 1:64. Antiserum C151 (anti-B.bovis Mexico isolate) had-. indirect fluorescent-antibody titers of1:5,120 and 1:640 against B. bovis and B. bigemina , respectively.Antiserum B85 immunoprecipitated eight [³⁵ S]methionine-radiolabledproteins of B. bovis. Four of the eight B. bovis proteinsimmunoprecipitated by B85 antiserum (120,000, 59,000, 53,000, and 19,000molecular weight) also had isolate-common epitopes. In addition, the120,000 molecular weight protein was one of the proteins recognized byC151 serum antibodies diluted 1:40.

EXAMPLE 9

Identification of Monoclonal Antibodies

Using similar techniques, additional MoAbs specific for surface-exposedepitopes on live merozoites were identified. All of the identified MoAbsare listed in Table 1. The MoAbs reacted with the outer surface ofculture- or stabilate-derived merozoites in either a punctate(restricted to a discrete region on the merozoite surface) or ahomogenous (over the entire surface of the merozoite) pattern whenexamined by IFA-live.

                  TABLE 1                                                         ______________________________________                                        Monoclonal antibodies generated against surface proteins                      on merozoites of Babesia bovis.                                                                     MW of Reactive                                                                Protein                                                 MoAb          Isotype (× 10.sup.-3 kd)                                  ______________________________________                                        23.8.34.24    G.sub.3 225                                                     BABB75        G.sub.2b                                                                              60                                                      MBOC79        G.sub.1 60                                                      23.53.156     G.sub.2a                                                                              60                                                      23.38.120.8   G.sub.1 60                                                      23.70.174.83  G.sub.1 44                                                      BABB35A.sub.4 G.sub.2a                                                                              42                                                      23.3.16       G.sub.1 42                                                      23.10.36      G.sub.2b                                                                              42                                                      23.28.57.108  G.sub.2a                                                                              16                                                      BABB90C.sub.4 G.sub.1 85                                                      BABB93A.sub.1 G.sub.2a                                                                              145                                                     ______________________________________                                    

All MoAbs reacted with merozoite antigen in dot immunoassay and alloweddetection of specific surface-exposed determinants in preparations of10⁵ lysed merozoites. The parasite specificity of these MoAb-reactivedeterminants was confirmed by immunoprecipitation ofmetabolically-radiolabeled parasite proteins of M_(r) 16 kDa, 42 kDa, 44kDa, 60 kDa, and 225 kDa. These proteins have been designated Bo16,Bo42, Bo44, Bo60, and Bo225, respectively. Bo225 was routinelyvisualized as a tightly spaced doublet when immunoprecipitated with MoAb23.8.34.24.

Seven monoclonal antibodies and the Mexico isolate of B. bovis have beendeposited with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852 USA. The cultures have beenassigned the following accession numbers by the repository:

    ______________________________________                                        Biological Material                                                                            Deposit number                                                                            Deposit date                                     ______________________________________                                        MoAb 23.38.120.8 HB 10111    May 2, 1989                                      MoAb BABB93A.sub.1                                                                             HB 10112    May 2, 1989                                      MoAb 23.8.34.24  HB 10113    May 2, 1989                                      MoAb 23.70.174.83                                                                              HB 10114    May 2, 1989                                      MoAb BABB35A.sub.4                                                                             HB 10115    May 2, 1989                                      MoAb 23.28.57.108                                                                              HB 10377    March 7, 1990                                    MoAb BABB90C.sub.4                                                                             HB10117     May 2, 1989                                      Babesia bovis, Mexico Isolate                                                                  ATCC 40601  May 3, 1989                                      ______________________________________                                    

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122.The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are fled. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent fights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

EXAMPLE 10

Construction of a Lambda-gt11 Expression Library

Partially purified and viable merozoites free from contaminating bovineleukocytes were obtained from frozen stabilates. Merozoites (1.4×10⁸6-CFDA-positive) were lysed in 10 mM Tris, 1 mM EDTA (TE buffer, pH 7.4)containing 2% SDS, and the suspension was treated with DNAse-free RNAseA (100 ug/ml) followed by Proteinase K (100 ug/ml). Genomic DNA wasisolated from the suspension by sequential phenol, phenol/chloroform,chloroform, and ether extractions followed by ethanol precipitation at0°-4° C. in the presence of 2M ammonium acetate. The DNA pellet waswashed once with 70% ethanol, lyophilized, resuspended in TE buffer andstored at 4° C. The concentration and purity of the DNA were assessed byspectrophotometry and agarose gel electrophoresis. The DNA was shearedinto fragments of between 4-8 kb by repeated passages through a 25 gaugehypodermic needle (Young, R. A., B. R. Bloom, C. M. Grosskinsky, J.Ivanyi, D. Thomas, and R. W. Davis [1985] Proc. Natl. Acad. Sci. USA82:2583-2587) and the fragments prepared for ligation into the EcoRIsite of the lambda-gt11 expression vector (Promega Biotec, Madison,Wis.).

First, fragments were methylated with EcoRI methylase (Promega Biotec)and blunt-ended using the large fragment of E. coli DNA polymerase I(Klenow Fragment, Bethesda Research Laboratories, Gaithersburg, Md.).EcoRI linkers (BRL, Gaithersburg, Md.), end-labeled with ³² Ptransferred from 5'-[gamma-³² P]ATP in a kinase reaction (Huyhn et al.,1985), were ligated to fragment termini using T4 DNA ligase (BethesdaResearch Laboratories). Free linkers were separated from fragments withEcoRI termini by size fractionation on a Sephacryl S-400 column(Pharmacia AB, Uppsala, Sweden) following digestion of the reactionmixture with EcoRI endonuclease.

Fractions containing fragments with EcoRI cohesive termini but free ofnonligated linkers were pooled, butanol-extracted to reduce volume,extracted twice with ether, ethanol-precipitated, lyophilized, andresuspended in TE buffer. Fragments were then ligated into the EcoRIsite of the lambda-gt11 expression vector which resulted in theinsertion of parasite DNA into the β-galactosidase structural gene(lacZ) of the bacteriophage (Young, R. A. and R. W. Davis [1983] Proc.Natl. Acad. Sci. USA 80:1194-1198). Ligated DNA was packaged into gammaphage heads (Gigapack Gold Packaging Extract, Stratagene CloningSystems, San Diego, Calif.) and the resultant library was amplified inE. coli strain Y1090 as described previously (Young and Davis, 1983).The amplified library was stored in sterile SM buffer (0.1M NaCl, 8 mMMgSO₄.7H₂ O, 50 mM Tris [pH 7.5], 2% gelatin) at 4° C.

EXAMPLE 11

Identification of Recombinant Phage Expressing Parasite Surface-ExposedProteins

Recombinant phage expressing proteins with surface-exposed epitopes wereidentified by immunoscreening plaques with MoAbs. Enough recombinantphage to give 10⁵ plaque forming units (pfu)/150 mm diameter petri dishwere used to infect E. coli host Y1090 by incubation at 37° C. for 20min in LB medium. Infected cells were added to LB top agar (55° C.)containing 100 ug/ml ampicillin and 10 mM MgCl₂ and plated out on 150 mmdiameter LB agar plates. Plates were incubated at 42° C. for 4 hr toallow plaque formation without concomitant expression of fusion protein.LacZ-directed gene expression was then switched on by overlaying eachplate with a dry nitrocellulose filter saturated previously with 10 mMIPTG and incubating the plates at 37° C. for 8-10 hr. After incubation,nitrocellulose filters with bound proteins were marked, removed from theplates, and processed as described previously for dot blot immunoassay.Single plaques expressing recombinant surface epitopes of interest wereidentified by autoradiography, picked from plates, and rescreened andpicked three more times to insure purity of the recombinant phage,stability of the DNA insert, and reliability of recombinant proteinexpression. Other hosts, such as Salmonella, can be transformed bysuitable procedures well known to those in the art.

Approximately 4.2×10⁶ recombinant plaques were screened with MoAbslisted in Table 1. Two recombinant clones (lambda-Bo44-15,lambda-Bo44-16) were identified that express a recombinant protein thatreacts with MoAb 23.70.174.83 (anti-Bo44) and two (lambda-Bo220-1 andlambda-Bo220-2) that express a recombinant protein that reacts with MoAb23.8.34.24 (anti-Bo225). When lambda-Bo44-15 and lambda-Bo44-16 weredigested with EcoRI, inserts (Bo44-15, Bo44-16) of 1.25 kb werevisualized for each recombinant clone. IPTG-induced lysogen preparationsof lambda-Bo44-15 consistently produced stronger signals in dot blotimmunoassay than did lambda-Bo44-16, and for this reason, lambda-Bo44-15was chosen for further analysis and use in immunization trials.

EXAMPLE 12

Induction of Recombinant Proteins with IPTG

E. coli host strain Y1089 was lysogenized with lambda-gt11 (control) andeach of the recombinant clones using standard procedures (Huyhn, T. V.,R. A. Young, and R. W. Davis [1985] "Constructing and Screening cDNALibraries in lambda-gt10 and lambda-gt11," In: DNA Cloning, Vol. 1: APractical Approach [Glover, D. M., ed.], pp. 49-78, IRL Press,Washington, D.C.). Lysogenized bacteria were examined by dot blotimmunoassay in order to determine the ability of clones to producerecombinant protein after induction with IPTG. Each of the recombinantclones, lambda-gt11-infected E. coli Y1089, and noninfected Y1089controls were grown at room temperature to OD₆₀₀ =0.8-1.2. At this time,an aliquot was removed, centrifuged, and lysed with lysis buffer (1%NP-40). The remaining cells were heated rapidly to 42°-45° C. for 20min, IPTG was added to 10 mM, and the cells were incubated in a shakingincubator at 37° C. for 1-2 hr to induce protein expression. Afterincubation, cell cultures were adjusted by addition of LB medium totheir OD₆₀₀ prior to addition of IPTG. At this time, an aliquot wasremoved, centrifuged, and the pellet lysed in lysis buffer. Three ulaliquots of pre- and post-induced bacterial lysates were spotted ontonitrocellulose in triplicate and probed with MoAbs specific for surfaceproteins or an irrelevant MoAb control (CAEV 4Al) in dot blotimmunoassays. Crude lysates of bacteria producing recombinant proteinwere prepared and stored at -20° C. or -70° C. until use.

Dot blot and Western blot analysis of lysates of bacteria lysogenizedwith lambda-Bo44-15 confirmed the inducibility of rBo44-15 with IPTG andits expression as a β-galactosidase fusion protein. rBo44-15 was visibleas a doublet of M_(r) 150 kDa and 165 kDa in Western blots ofIPTG-induced preparations probed with MoAb 23.70.174.83. In contrast,rBo44-15 was visible as a single band (165 kDa) in Western blots ofidentical antigen preparations probed with anti-β-galactosidase. Westernblots of lambda-Bo44-15 lysogen preparations probed with an irrelevantIgG₁ control MoAb (5.90.1) showed no reactivity, thus confirming thespecificity of reaction observed with MoAb 23.70.174.83.

EXAMPLE 13

Purification of Recombinant Proteins

MoAb 23.70.174.83 was purified from ascitic fluid by ammonium sulfateprecipitation and DEAE cellulose chromatography and then coupled toSepharose 4B for immunoaffinity purification of rBo44-15. Solubilizedand sonicated rBo44-15 lysogen preparations were applied to the affinitycolumn, the column was washed repeatedly, then adherent recombinantprotein was eluted with 0.1M diethylamine (pH 11.5) containing 0.5%deoxycholate. Elutes were collected directly into 1M Tris (pH 8.5) thendialyzed against PBS to remove detergents. Aliquots of partiallypurified protein preparations were boiled for 10 min in SDS samplebuffer, subjected to SDS-PAGE, silver stained and examined by Westernblot immunoassay to verify the presence and purity of recombinantprotein. Total protein concentration of the preparations was determinedusing a bicinchoninic acid protein assay (Pierce Chemical Co.).

Partially purified rBo44-15 from affinity column chromatographycontained several high M_(r) proteins ranging from approximately 94 Kdto >165 Kd as well as several lower M_(r) proteins ranging from 26 Kd to50 Kd. Western blot analysis of column-purified protein preparationsrevealed two major bands of reactivity at M_(r) 165 Kd and 150 Kd thatcorrespond to two major bands present in silver-stained gels. Inaddition, several bands of lower M_(r) (26-31 Kd) were observed inWestern blots of affinity-purified recombinant protein that were notobserved in Western blots of solubilized lysogen preparations prior toaffinity purification. These data indicate that immunoaffinitychromatography results in partial degradation of the recombinantmolecule without a concomitant loss of integrity of the MoAb-reactiveepitope.

EXAMPLE 14

Confirmation of the Presence of Surface-Exposed Epitopes on RecombinantMolecules

Five Holstein-Freisian bull calves were each immunized intramuscularly(i.m.) with 100-125 ug of affinity column-purified recombinant proteinin Freund's complete adjuvant, followed by four to five additionalimmunizations at three week intervals of recombinant protein in Freund'sincomplete adjuvant. Control calves were immunized similarly with 100 ugovalbumin. Within one week after the last immunization, calves were bledand their sera heat-inactivated and examined by IFA-live (Goff et al.,1988) to confirm the presence of antibodies to surface-exposed epitopeson merozoites. Hyperimmune bovine serum (KLK C151) and preimmune serawere used as positive and negative controls, respectively. Sera fromcalves immunized with either rBo44-15 or ovalbumin were used toimmunoprecipitate metabolically radiolabeled merozoited proteins(McElwain et al., 1987) in order to verify the specificity of theantibody response.

Antibody titers in serum from calves immunized with partially purifiedrBo44-15 varied from 10⁻² to 10⁻⁵ as evidenced by IFA-live. In contrast,preimmune serum (B452-pre) and serum from ovalbumin-immunized calvesshowed reactivity with live merozoites at dilutions of 10⁻¹ and 10⁻²,respectively. Antibody in all nondiluted sera or in low serum dilutions(10⁻¹, 10⁻²) bound to erythrocyte ghosts (infected and noninfected) aswell as merozoites within ghosts.

EXAMPLE 15

Methods and Materials for Construction of DNA Probe

(a) Parasites and DNA Isolation

Strains of parasites used in this study include a Mexico (M) andAustralia (S strain) isolate of B. bovis and a Mexico isolate of B.bigemina. Babesia bovis (M) DNA for both the genomic library preparationand analysis of clones was derived from infected bovine erythrocytecultures washed three times in phosphate buffered saline (PBS), pH 7.2,followed each time by centrifugation at 400×g. Babesia bovis (S) and B.bigemina DNA was similarly derived from infected calf blood depleted ofbuffy coats by three washes in PBS. Infected erythrocytes for isolateswere differentially lysed in nine volumes of 0.42% NaCl, infected ghostswere pelleted at 400×g, lysed in 5 volumes of 10 mM Tris-HCl (pH 7.5),10 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, and 1% sodiumdodecyl sulfate (SDS), incubated 16 hr with proteinase K (100 ug/ml),extracted with phenol:chloroform:isoamyl alcohol. (24:24:1). DNA in theaqueous phase was spooled after addition of 2 volumes of cold ethanol,spooled DNA was dried and resuspended in 10 mM Tris-HCl (pH 7.4) and 1mM EDTA (TE), treated with RNases A and Tl (15 ug/ml, 15 units/ml,respectively). The solution was reextracted, spooled, dried, andresuspended in TE for use.

To obtain bovine leukocyte DNA, cells in the buffy coat of uninfectedblood were processed similar to infected erythrocytes.

(b) Identification and Isolation of Recombinant B. bovis DNA

The preparation of the genomic library has been described. Briefly, B.bovis genomic DNA was sheared through a 26 gauge needle to sizes rangingfrom c4-8 Kb, methylated with EcoRI methylase, EcoRI linkers were added,DNA restricted with EcoRI and separated from digested linkers onsephacryl S-400 (Pharmacia), and DNA ligated into EcoRI digested anddephosphorylated arms of lambda-gt11. Recombinant phage were amplifiedin Escherichia coli strain Y1089 (Stratagene).

DNA from phage plaques was adsorbed onto nitrocellulose and replicatefilters were differentially hybridized to nick translated DNA (2×10⁶cpm/ml) from either B. bovis (M) or B. bigemina in 6× SSPE (1× SSPE:150mM NaCl, 10 mM NaH₂ PO₄, 1 mM EDTA, pH 7.4), 100 ug/ml denatured salmonsperm DNA, and 1% SDS at 65° C. for 16 hr. Filters were washed twice in2× SSPE and 1% SDS at room temperature for 20 min, and twice in 0.1×SSPE and 0.1% SDS at 65° C. for 20 min. Dried filters wereautoradiographed at -70° C. Recombinant phage hybridizing to B. bovis(M) but not detectably to B. bigemina DNA were purified through 3 roundsof rescreening and isolated for further analysis. Based oncharacteristics of hybridization to B. bovis (M) genomic DNA, insertsfrom some recombinants were cloned into plasmid pBS⁺ (Stratagene) tofacilitate their analysis.

(c) Southern and Dot Blot Assays

To investigate the genomic organization of candidate probe sequences,restriction fragments were separated electrophoretically on 0.7% agarosegels and transferred to nylon filters. Filters were then hybridized, asdescribed above, in the presence of 10% dextran sulfate, to lambda-gt11recombinant DNA or preparatively isolated insert DNA that wasradioactively labeled. Final wash strigency was either 65° C. or 50° C.,as indicated.

For dot blot analysis, DNA extracted as described above was spotted ontonylon membranes. Membranes were dried at room temperature, saturatedwith 0.5 M NaOH and 1.5M NaCl, neutralized in 1M ammonium acetate and0.02M NaOH, rinsed in 6× SSPE, and vacuum-dried at 80° C. for 1 hr.Hybridization conditions were similar to those used for Southern blots.Sensitivity of probe sequences was determined for autoradiograms exposedto hybridization filters for approximately 16 hr at -70° C. using anintensifying screen.

EXAMPLE 16

Candidate DNA Probes for Detecting Babesia bovis in Infected Ticks andCattle

DNA-DNA hybridization assays (DNA probes) are based on the fact thatsingle-stranded DNA will reanneal only with a complementary strand ofDNA whose sequence is homologous. DNA probes have been used as a meansof detecting various infectious agents, and some are now used routinelyin clinical microbiology laboratories. The identification of DNAsequences of Babesia spp. makes it possible to create DNA probes for theidentification of these species. Therefore, one application of theidentification and isolation of genomic sequences which encode babesialantigens is the use of the DNA fragments as DNA probes.

The lambda-gt11 genomic DNA library of Babesia bovis was screened toidentify DNA probe candidates for direct detection of the parasite inblood or ticks infected with the parasite. Two sequences (lambda-Bo6 andlambda-Bo25) demonstrated superior sensitivity and were analyzed in moredetail. The insert size of lambda-Bo6 is 2.75 kilobase pairs (kb). Anaccurate estimate of the lambda-Bo25 insert was not possible since oneof the EcoRI insert sites was lost during cloning. However, digestion oflambda-Bo25 with EcoRI and KpnI produces a fragment of 2.2 kb which isspecific to this clone compared to wild type lambda-gt11. Since the KpnIsite in lambda-gt11 occurs approximately 1 kb from the EcoRI cloningsite, a minimum estimated size of the insert is 1.2 kb.

Inserts from the lambda clones were excised with EcoRI. (lambda-Bo6) orSstI and KpnI (lambda-Bo25) and cloned into the plasmid BS⁺(Stratagene), producing the two clones pBo6 and pBo25. Insert fragmentspreparatively isolated from these plasmid clones were radioactivelylabeled and used in dot and Southern blot analyses.

Neither sequence hybridized detectably to bovine DNA. pBo6 detected 100pg of both a Mexico and an Australia isolate of B. bovis, but pBo6 alsodetects 1.0 ng of B. bigemina DNA under identical conditions. A uniquecharacteristic of pBo6 is that it hybridizes to a 7.4 kilobase band inuncut genomic DNA of both B. bovis and B. bigemina. Similarity ofrestriction enzyme patterns of the pBo6 sequence in genomic DNA fromboth geographic isolates suggests that this sequence is well conservedbetween geographic isolates of B. bovis. Thus, this sequence is acandidate DNA probe for detecting B. bovis infections in cattle andticks.

pBo25 exhibited no detectable hybridization to bovine or B. bigeminaDNA. This sequence detected 100 pg of homologous Mexico isolate DNA, butunder identical conditions the sensitivity was reduced to 1 ng forAustralia isolate DNA. Restriction enzyme analysis of the pBo25 sequenceshowed major differences in the number, size, and intensity of bandsbetween the two B. bovis geographic isolates tested. Thus, this sequencecan distinguish geographic isolates of B. bovis.

EXAMPLE 17

Labeling of DNA Probe

In order to facilitate detection, DNA probes can be labeled in a varietyof ways. For example, for biotin labelling, the DNA fragment preparationis adjusted to a concentration of 1 mg/ml (TE) and is mixed withphoto-activatable biotin (PAB) at a ratio of 1:3 (DNA:PAB) in a 1.5 mlEppendorf tube. The tube is placed in an ice bath 10 cm below a 275 W(GE RSM) sunlamp and the DNA+PAB is irradiated for 15 min. The DNAsolution is then mixed with an equal volume of 0.1M Tris-Cl (pH 9.0) andthe volume adjusted to 100 ul with H₂ O. The unincorporated PAB isextracted from the DNA by the addition of an equal volume of 2-butanol,vortexing, centrifuging briefly, and withdrawing the lower aqueous phasewith a Pipetman. The extraction can be repeated to remove any traces ofunbound PAB. 3M NaOAc (pH 5.6) is added to the DNA solution to a finalconcentration of 0.3M and the labeled DNA is precipitated by theaddition of 3 volumes of ethanol.

After the sample is cooled at -70° C. for 15 min, the precipitated DNAis recovered by centrifugation for 10 min. The DNA pellet is dissolvedin 10 mM Tris (pH 7.9) and 0.1 mM EDTA. The labeled probe DNA remainsstable for 1 year if stored at -20° C.

A non-radioactive method of labeling the DNA probes may be desirablebecause: 1) the photoactivatable reactions are simple and rapid, 2) thesensitivity is as high as ³² P-labeled probes, 3) the PAB-labeled probeshave a long storage life, 4) these probes are relatively inexpensive,and 5) detection of bound probes is by simple colorimetric methods. Theradioactive labeling of probes requires the use of 32P, which has a veryshort half-life (14 days) and is thus unstable and expensive. The use ofradioactive probes would be limited because of cost, the dangers ofradioactivity, strict requirements for disposal, and the need forlicensing.

However, if for some reason the biotin-HRP method of labeling is notacceptable, the DNA fragments can be labeled with [gamma-P] 32 deoxy CTPby standard nick translation methods.

EXAMPLE 18

Description of Recombinant DNA Sequence from B. bovis that Encodes anImmunoreactive Epitope Located on the Surface of Merozoites

The cloned insert DNA was excised from the lambda-gt11 vector andrecloned into the plasmid BS⁺ (Stratagene), producing the clonepBo44-15. DNA templates for sequencing the insert of pBo44-15 wereobtained by creating deletion libraries of this clone using exonucleaseIII and mungbean nuclease. A different deletion library was obtainedstarting at each end of the clone, which allowed sequencing of bothstrands of the insert DNA. The DNA sequence was obtained using theSanger dideoxy method.

The sequence of Bo44-15 insert DNA is shown in FIG. 1. The insert is1235 base pairs long. The amino acid sequence shown represents the onelong open reading frame identified in the sequence. The open readingframe begins at position 1 and encodes a stop codon (TAA) beginning atposition 568. This reading frame is in correct register for expressionas a fusion protein of β-galactosidase in lambda-gt11, provided theclone is in the correct orientation. A notable feature of this openreading frame is that it would encode a 24 amino acid sequence beginningat AA position 85 which is tandemly repeated beginning at AA position109. Comparison of these two putative repeats shows only two positionsthat differ between the repeats as shown below:

    ______________________________________                                         85     PQRPAETQQTQDSAAPSTPAAPSP                                                                             108                                            109                                                                                    ##STR1##              132                                            ______________________________________                                    

Numbers represent the beginning and ending amino acid position in theopen reading frame for each repeat. Letters are the single letter codefor amino acids. Asterisks below the aligned repeats indicate amino aciddifferences between the two repeats.

The potential significance of the repeat amino acid sequence is thatsuch repeats are often immunodominant epitopes in surface proteins froma variety of other protozoan parasites, and they induce antibodies thatprotect against the diseases.

The DNA sequences coding for two other of the B. bovis proteins havealso been discovered. The DNA sequence for the B. bovis surface proteinsof 42 kDA and 60 kDA are shown in FIGS. 2 and 3, respectively. Thesesequences, or portions of the sequences, can be used as DNA probes asdescribed in Examples 16 and 17. Also, the proteins produced from cellstransformed with these sequences, or portions of these sequences, can beused for vaccines or in the preparation of monoclonal antibodies asdescribed in the examples which follow.

The procedure for obtaining these sequences are described below:

B. bovis cDNA Expression Library. Erythrocytes from asynchronous B.bovis-infected blood cultures were washed three times in Puck's saline Gand stored frozen in liquid nitrogen. Cells were thawed in lysis buffercontaining 0.2M NaCl, 0.2M Tris-HCl pH 7.5, 1.5 mM MgCl₂, 2% SDS (w/v),and 200 μg/ml Proteinase K and then incubated in lysis buffer at 46° C.for two hours. (Bradley, J. E., G. A. Bishop, T. St. John, and J. A.Frelinger [1988] Biotechniques 6:114-116). The NaCl concentration of thelysate was adjusted to 0.5M and poly [A⁺ ] RNA was isolated by batchadsorption with oligo(dT) cellulose (Bradley et al., supra). DNA elutedwith 0.01M Tris-HCl pH 7.5 was used to prepare a blood stage cDNAlibrary in Lambda ZAP II (Stratagene, La Jolla, Calif., USA) (Short, J.M., J. M. Fernandez, J. A. Sorge, and W. D. Huse [1988] Nucl. Acids Res.16:7583-7600) by a modified Gubler and Hoffman method using EcoRIadapters (Pharmacia LKB, Piscataway, N.J., USA) (Gubler, U., and B.Hoffman [1983] Gene 25:263-269). The cloned insert in plaque purifiedlambda phage was subcloned into Bluescript SK(-) phagemid using the invivo excision capabilities of Lambda ZAP II (Short et al., supra).

Immunoscreening. Plaque lifts ont isopropyl thiogalacto-pyranosidesoaked nitrocellulose were screened using monospecific rabbit anti-Bv42antisera (R-914) followed by ¹²⁵ I-Protein A and autoradiography, orwith anti-Bv60 monoclonal antibody (23.38.120.8) followed by rabbitanti-murine immunoglobulin, ¹²⁵ I-Protein A and autoradiography (Young,R. A., and R. W. Davis [1983] Proc. Natl. Acad. Sci. USA 80:1194-1198;Reducker, D. W., D. P. Jasmer, W. L. Goff, L. E. Perryman, W. C. Davis,and T. C. McGuire [1989] Mol. Biochem. Parasitol. 35:239-248). RabbitR-914 had been immunized with native By42 protein immunoaffinitypurified using BABB35A₄, a previously described monoclonal antibody(Goff, W. L., W. C. Davis, G. H. Palmer, T. F. McElwain, W. C. Johnson,J. F. Bailey, and T. C. McGuire [1988] Infect. Immun. 56:2363-2368).Positive Bv42 plaques were tested for reactivity with monoclonalantibodies that recognize a Bv42 surface exposed epitope (Goff et al.,supra; Reducker et al., supra) as well as an isotype control monoclonalantibody and normal rabbit serum. Recombinant phagemid excised frompositive, plaque purified lambda phage was tested for expression by asimilar method using colony lifts from transformed, ampicillin resistantE. coli (XL1-Blue strain) (Young and Davis, supra).

Restriction Enzyme Digestion. Lambda rBv42 phagemid DNA was isolatedfrom bacteria by anion exchange chromatography (Qiagen Inc., StudioCity, Calif.) and restriction enzyme digested by standard methods(Maniatis, T., E. F. Fritsch, and J. Sambrook [1982] Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 545 pp.).

EXAMPLE 19

Vaccines

Vaccines may be produced from the polypeptides expressed by theparasites themselves or by cells which have been transformed with DNAfragments from Babesia. By introducing these polypeptides, along with apharmacologically suitable vehicle or adjuvant, into the animal host,that host can be induced to generate immunological protection againstBabesia. The preparation of such a vaccine composition is within theskill of one trained in the medical and immunological sciences. Vaccinesmay utilize entire polypeptides or epitopes with immunological activity.

EXAMPLE 20

Monoclonal Antibodies

Appropriate mice can be immunized with antigens of, or cells expressingantigens of, Babesia. The antigens used for this immunization can bethose which are identified and described in the previous examples. Thetechniques employed to accomplish this immunization procedure arefamiliar to those skilled in this art. The spleens can then be removedfrom the immunized mice and the cells therefrom fused to SP-2 myelomacells using polyethylene glycol. The desired hybrid cells can then beselected by adding hypozanthine-aminopterin-thymidine to the medium. Thesurviving cells can then be tested for antibody production. The testingfor antibody production can be accomplished using IFA, ELISA,immunoblot, and/or immunoprecipitation procedures.

EXAMPLE 21

Detection of Babesia Antigens

The monoclonal antibodies, such as those produced by the procedure justdescribed or those disclosed in Examples 1 and 9, can be used to testfor the presence of Babesia antigens in a sample of biological fluid.Other monoclonal antibodies to Babesia antigens can also be used. Thetesting procedure involves contacting the biological fluid with acomposition containing one or more of the monoclonal antibodies. IfBabesia antigens are present in the biological fluid, then a reactionwill occur and this reaction can be detected and quantified byfluorescence or other means.

EXAMPLE 22

Detection of Anti-Babesia Antibodies

Anti-Babesia antibodies can be detected in a fluid sample from a bovinesuspected of containing these antibodies by performing ELISA procedureson the clinical samples. Generalized ELISA procedures are well known tothose skilled in the art. The ELISA procedures or other simplediagnostic procedures of the subject invention could utilize asantigens, for example, whole cell or cell lysate using recombinantmicroorganisms which express Babesia antigens.

When the biological sample is contacted with the whole cell or celllysate microorganisms, this contacting is done under conditions whichwill promote antigen/antibody immunocomplex formation between antigensexpressed by the microorganism and antibodies present in the sample. Theresulting immunocomplex can be readily detected utilizing standardlabeling procedures.

EXAMPLE 23

Biological Deposit Information

Biological material which can be used according to the subject inventionhas been deposited in the permanent collection of the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852USA. The accession number and deposit date are as follows:

    ______________________________________                                        Depositor Identification                                                                     Repository No.                                                                             Deposit Date                                      ______________________________________                                        Plasmid, pBo6  75575        October 19, 1993                                  ______________________________________                                    

The subject DNA clone has been deposited under conditions that assurethat access to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. Thedeposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent fights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. Any restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

We claim:
 1. A microorganism transformed with lambda-Bo6 or plasmidpBo6.
 2. An isolated DNA sequence consisting of the 2.75 kb EcoRIfragment of plasmid pBo6.